Project Summary Aggressive metastatic breast cancer is responsible for the deaths of more than 40,000 people per year in the U.S., despite the best efforts of researchers and clinical oncologists. I have recently discovered a potentially important mechanism regarding the generation of a unique secretome by breast cancer cells, which makes an essential contribution to their invasiveness and metastatic capability. Specifically, I have delineated an intriguing connection between the down-regulation of Sirtuin 1 (Sirt1), a NAD-dependent deacetylase, in aggressive breast cancer cells and the corresponding reduction in the expression of a major subunit of the vacuolar ATPase (V-ATPase), which results in the impairment of their lysosomes and consequently, dramatic changes in their secretome. These changes include a significant increase in the number of exosomes generated by breast cancer cells, and an enrichment in their ubiquitylated protein cargo. Exosomes are small (extracellular) vesicles, ~30-150 nm in size, that contain a wide range of cargo including proteins, RNA transcripts, microRNA, and even DNA. They function as mediators of intercellular communication and have been implicated in a number of aspects of cancer progression, including the promotion of chemo-resistance and the formation of a pre-metastatic niche. Because exosomes are also attractive vehicles for the delivery of therapeutic agents, studies aimed at determining how exosomes are formed and released, as well as characterizing their functional properties, are being extensively pursued. Thus, these findings now highlight how aggressive breast cancer cells generate exosomes containing unique cargo, which contribute to the metastatic capability of breast cancer cells. I further discovered that the down-regulation of Sirt1 in breast cancer cells results in a significant increase in the secretion of soluble hydrolases, in particular, cathepsins. Collectively, these components making up the secretome of aggressive breast cancer cells give rise to a marked enhancement in migratory and invasive activity. In the F99 phase of this application, these discoveries will be extended by determining the underlying mechanisms by which the down-regulation of Sirt1 in aggressive cancer cells leads to a reduced expression of the V-ATPase (Aim 1). A particular emphasis will be to identify the Sirt1 substrate that is directly responsible for regulating the stability of the RNA transcript encoding one of the major subunits of the V-ATPase. In the K00 phase of the proposal (Aim 2), a research/training environment will be sought to develop animal models that will further establish the functional connection between Sirt1, the v-ATPase and lysosomal function, and demonstrate how this contributes to the metastatic state. The ultimate goal of my studies will be to highlight strategies that alter these connections in a manner that leads to new anti-cancer therapies.